Throughout this application various publications are referred to in parenthesis. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Fenretinide and its Significance in Clinical and Biological Studies:
4-HPR [N-(4-hydroxyphenyl)retinamide, or Fenretinide] is a synthetic retinoid that has potent chemopreventive and antiprolliferative effects against many cancers in vitro and in preclinical models, and it does not show appreciable side effects. It exhibits cytotoxicity and in vitro suppresses tumor cell growth at low micromolar concentrations (IC50s) ranging from 1-10 μM (1). 4-HPR is an FDA approved drug under phase II clinical trials for many cancers including neuroblastomas, currently sponsored by the National Cancer Institute (Ref: 06-C-0227). Fenretinide has been largely studied as a chemo-preventive agent in carcinogen-induced epithelial tumors in animal models and in patients at risk for breast cancer (2-5). In advanced metastatic breast cancer, 4HPR has minimal activity; however, in comparison to other retinoids, has low toxicity (2-4). However, recent results of a fifteen-year follow up studies of Phase III trials of Fenretinide to prevent second breast cancer indicated that it has significant risk reduction in premenopausal women, which is remarkable at younger age, and persists after several years (6). These studies indicate that Fenretinide has promising preventive activity in clinical trials of breast cancer. In pediatric neuroblastoma patients, use of 4HPR has demonstrated prolonged stabilization of disease in pilot clinical studies (7-10).
4HPR induces apoptosis in tumor cell lines in vitro by various mechanisms including: (i) activation of retinoid receptors RAR β and γ; (ii) induction of ceramide-dependent cell cytotoxicity that is independent of p53 or caspase-3 function and thus is synergistic with tamoxifen, which in turn is an inhibitor of glucosylceramide synthase; (iii) generation of free radical oxygen species; (iv) increase of NOS expression resulting in increased NO-dependent cell cytotoxicity; and (v) increase of mitochondrial permeability transition (2, 4, 7, 8, 10). 4HPR also induces cell cycle arrest and down modulates the expression or activity of proliferation related targets such as c-myc, telomerase, p34/cdc2 and Cyclin (10-21). These effects correlate with the induction of phosphorylation of Rb, cell cycle arrest and subsequent induction of apoptosis.
Over-expression of CyclinD1 is sufficient to sensitize certain cancer cells to 4-HPR, indicating that CyclinD1 may be a key cellular target for the action of this drug (22). Consistent with this idea, 4-HPR appears to affect the expression as well as the protein stability of cyclin D1 in a concentration dependent manner (23-26), and in leukemia cell lines, efficacy of 4HPR correlates with CyclinD1 depletion.
This information about the ability of Fenretinide to target Cyclin D1 was used to inhibit rhabdoid tumors, which are highly malignant pediatric tumors (27). Previous studies conducted at Albert Einstein College of Medicine indicated that rhabdoid cells over-express CyclinD1, and that these cells are critically dependent on Cyclin D1, using both in vitro cell culture models and in vivo genetically engineered mouse (GEM) models of rhabdoid tumors that are heterozygous for Ini1 locus (28-30). Based on these studies it was surmised that rhabdoid cells might be sensitive to 4HPR. Consistent with this hypothesis, it was established that rhabdoid tumors indeed are sensitive to 4-HPR in vitro and in vivo and its effect is correlated to down-modulation of Cyclin D1 (28).
4-HPR as a Therapeutic Agent for Diseases Other than Cancer:
4-HPR is a retinoid and as such it is likely to interfere with the retinoic acid pathway in the cells and affect the biology of the pathway. Since defects in retinoic acid synthesis, metabolism, and transcriptional regulation of downstream genes by its ability to bind to nuclear receptors (RAR and RXR) are important for growth, development, behaviour, and disease pathways (31-37), retinoic acid metabolism inhibitors are widely used as therapeutic agents in many diseases (38).
Fenretinide is a synthetic retinoid that induces apoptosis in cancer cells as opposed to retinoic acid and other retinoids that induce differentiation (4). Because of this reason, and because of its low toxicity and effect on many different pathways including ceramide biosyntesis, free radical oxygen, and NOS, Fenretinide has been widely investigated as a preventive or therapeutic agent in many diseases. In addition to cancer, several preclinical studies have suggested activity of this compound against an array of diseases including but not limited to diabetes, AIDS, Alzheimer's Disease, cystic fibrosis, allergic encephalomyelitis, and ichthyosis (4, 39-61).
4-HPR Analogues in Inducing Cytotoxicity:
4-HPR is one of the most widely investigated synthetic retinoids for cancer prevention, especially for breast cancer. Pharmacological studies in human clinical trials of breast cancer patients have revealed accumulation of plasma concentration of 4-HPR at 1 μM levels with administration of 200 mg/day (MTD) of 4-HPR (3). It is possible that a molar concentration higher than currently attainable within the tumors may be required to achieve desired cytocidal effect with 4-HPR in other human cancers. Additional studies have indicated that in vitro activity of Fenretinide does not match a correspondent efficacy in vivo, indicating a need for further improvement of the drug. Many reasons has been proposed to explain the discrepancy between in vitro and in vivo activities of the drug, including decreased bioavailability and inability of the drug to cross the blood-brain barrier. The lack of bioavailability could be due to the hydrophobicity of the drug, where it never reaches amounts suitable for therapeutic response within the tumors. One report indicated the development of 4-HPR linked to polyvinylalcohol (PVA) (62). These studies indicated the feasibility of linking Fenretinide to improve its bioavailability.
There are few studies where synthetic analogues of 4-HPR have been reported to be active in cell toxicity studies. For example, it was reported that a non-hydrolysable carbon linked analogue of 4-HPR (N-benzyl hydroxyl retinamide, 4-HBR), potentially reduces suppression of plasma vitamin A levels (63, 64). The sulfur-containing heteroretinoids induce apoptosis and reactive oxygen species specifically in malignant but not in benign cells (65). Conjugations of 4-HPR also have been reported to be effective in antitumor activity. It was reported that anti-tumor potency of 4-HPR increases when it was conjugated to glucuronides. Glycosyl conjugated mannosyl with 4-HPR increased activity on promyelocytic leukemia cell lines HL60 (66). Recently, it was reported that 4-Oxo-fenretinide induced marked G2-M cell cycle arrest and apoptosis in fenretinide-sensitive and fenretinide resistant cell lines (67). Thus, there continues to be a need for improved 4-HPR derivates that demonstrate more potent biological activity efficacy and improved bioavailability and ability to cross the blood brain-barrier compared to parent 4-HPR.